Project Summary Regulated cell adhesion and migration are critical for a variety of biological processes including early embryonic development, neuronal path-finding, inflammatory response, wound healing and tissue integrity, cell-mediated immunity, and tumor invasion and metastasis. Integrin trans-membrane receptors are key regulators of cell migration, and act through an intricate assembly of regulatory proteins associated with the integrin cytoplasmic domain, many of which function as molecular scaffolds. The goal of this research is to identify novel regulatory pathways by which scaffolding proteins modulate cell adhesion and migration via regulated binding of protein partners. In previous work, we characterized the LIM domain scaffold PINCH, which is required for integrin-dependent cell adhesion, migration and signaling. PINCH binds with high affinity to both Integrin-linked kinase (ILK) and Ras suppressor 1 (RSU1), a Leucine-rich repeat protein that suppresses transformation of cultured cells expressing activated Ras, an oncogene present in 30% of all human tumors. We propose that the PINCH scaffold serves as a node for modulating integrin signaling by regulated recruitment of partners like ILK and RSU1, coupled to other signaling pathways. Through a series of experiments in Drosophila in which we specifically disrupt binding interactions with PINCH and alter the subcellular localization of components of the PINCH complex, we will dissect the requirement for ILK and RSU1 binding in carrying out the integrin-dependent functions of PINCH. Our preliminary data indicates that binding to ILK localizes PINCH within integrin-associated complexes at the cell membrane, and regulates the shuttling of PINCH to the nucleus. We will determine if PINCH has essential functions in the nucleus, and whether it has an overlapping complement of binding partners in the cytosol versus the nucleus. Additional data suggest that RSU1 is sequestered in integrin-associated complexes by its interaction with PINCH. We will examine how PINCH-RSU1 binding is regulated, and identify partners and activities for RSU1 that are independent of PINCH. A molecular understanding of how PINCH regulates cell adhesion and migration through its scaffolding activity may ultimately suggest strategies for prevention and therapeutic intervention in human pathologies like metastatic cancer, in which aberrant cell migration plays a prominent role.